Kit-positive interstitial cells in the rabbit urethra: structural relationships with nerves and smooth muscle.

OBJECTIVE: To identify interstitial cells (ICs) in the wall of the rabbit urethra using antibodies to the Kit receptor, and to examine their location, morphology and relationship with nerves and smooth muscle cells (SMCs), as studies of enzymatically isolated cells from the rabbit urethra have established that there are specialized cells that show spontaneous electrical activity and have morphological properties of ICs. MATERIALS AND METHODS: Urethral tissues from rabbits were fixed, labelled with antibodies and examined with confocal microscopy. Some specimens were embedded in paraffin wax and processed for histology. Histological sections from the most proximal third and mid-third region of rabbit urethra were stained with Masson's Trichrome to show their cellular arrangement. RESULTS: Sections from both regions had outer longitudinal and inner circular layers of SM, and a lamina propria containing connective tissue and blood vessels; the lumen was lined with urothelial cells. The mid-third region had a more developed circular SM layer than the most-proximal samples, and had extensive inner longitudinal SM bundles in the lamina propria. Labelling with anti-Kit revealed immunopositive cells within the wall of the rabbit urethra, in the circular and longitudinal layers of the muscularis. Double-labelling with an antibody to SM myosin showed Kit-positive cells on the boundary of the SM bundles, orientated parallel to the axis of the bundles. Others were in spaces between the bundles and often made contact with each other. Kit-positive cells were either elongated, with several lateral branches, or stellate with branches coming from a central soma. Similar cells could be labelled with vimentin antibodies. Their relationship with intramural nerves was examined by double immunostaining with an anti-neurofilament antibody. There were frequent points of contact between Kit-positive cells and nerves, with similar findings in specimens double-immunostained with anti-neuronal nitric oxide synthase (nNOS). CONCLUSION: Kit-positive ICs were found within the SM layers of the rabbit urethra, in association with nerves, on the edge of SM bundles and in the interbundle spaces. The contact with nNOS-containing neurones might imply participation in the nitrergic inhibitory neurotransmission of the urethra. PMID: 17212607 [PubMed - indexed for MEDLINE]

A comparison of conformal and intensity-modulated techniques for oesophageal radiotherapy.

Background and purpose: To investigate the potential of intensity-modulated radiotherapy (IMRT) to reduce lung irradiation in the treatment of oesophageal carcinoma with radical radiotherapy.Materials and methods: A treatment planning study was performed to compare two-phase conformal radiotherapy (CFRT) with IMRT in five patients. The CFRT plans consisted of anterior, posterior and bilateral posterior oblique fields, while the IMRT plans consisted of either nine equispaced fields (9F), or four fields (4F) with orientations equal to the CFRT plans. IMRT plans with seven, five or three equispaced fields were also investigated in one patient. Treatment plans were compared using dose-volume histograms and normal tissue complication probabilities.Results: The 9F IMRT plan was unable to improve on the homogeneity of dose to the planning target volume (PTV), compared with the CFRT plan (dose range, 16.9+/-4.5 (1 SD) vs. 12.4+/-3.9%; P=0.06). Similarly, the 9F IMRT plan was unable to reduce the mean lung dose (11.7+/-3.2 vs. 11.0+/-2.9 Gy; P=0.2). Similar results were obtained for seven, five and three equispaced fields in the single patient studied. The 4F IMRT plan provided comparable PTV dose homogeneity with the CFRT plan (11.8+/-3.3 vs. 12.4+/-3.9%; P=0.6), with reduced mean lung dose (9.5+/-2.3 vs 11.0+/-2.9 Gy; P=0.001).Conclusions: IMRT using nine equispaced fields provided no improvement over CFRT. This was because the larger number of fields in the IMRT plan distributed a low dose over the entire lung. In contrast, IMRT using four fields equal to the CFRT fields offered an improvement in lung sparing. Thus, IMRT with a few carefully chosen field directions may lead to a modest reduction in pneumonitis, or allow tumour dose escalation within the currently accepted lung toxicity.

Optimisation of radiotherapy for carcinoma of the parotid gland: a comparison of conventional, three-dimensional conformal, and intensity-modulated techniques

Background and purpose: To compare external beam radiotherapy techniques for parotid gland tumours using conventional radiotherapy (RT), three-dimensional conformal radiotherapy (3DCRT), and intensity-modulated radiotherapy (IMRT). To optimise the IMRT techniques, and to produce an IMRT class solution.Materials and methods: The planning target volume (PTV), contra-lateral parotid gland, oral cavity, brain-stem, brain and cochlea were outlined on CT planning scans of six patients with parotid gland tumours. Optimised conventional RT and 3DCRT plans were created and compared with inverse-planned IMRT dose distributions using dose-volume histograms. The aim was to reduce the radiation dose to organs at risk and improve the PTV dose distribution. A beam-direction optimisation algorithm was used to improve the dose distribution of the IMRT plans, and a class solution for parotid gland IMRT was investigated.Results: 3DCRT plans produced an equivalent PTV irradiation and reduced the dose to the cochlea, oral cavity, brain, and other normal tissues compared with conventional RT. IMRT further reduced the radiation dose to the cochlea and oral cavity compared with 3DCRT. For nine- and seven-field IMRT techniques, there was an increase in low-dose radiation to non-target tissue and the contra-lateral parotid gland. IMRT plans produced using three to five optimised intensity-modulated beam directions maintained the advantages of the more complex IMRT plans, and reduced the contra-lateral parotid gland dose to acceptable levels. Three- and four-field non-coplanar beam arrangements increased the volume of brain irradiated, and increased PTV dose inhomogeneity. A four-field class solution consisting of paired ipsilateral coplanar anterior and posterior oblique beams (15, 45, 145 and 170o from the anterior plane) was developed which maintained the benefits without the complexity of individual patient optimisation.Conclusions: For patients with parotid gland tumours, reduction in the radiation dose to critical normal tissues was demonstrated with 3DCRT compared with conventional RT. IMRT produced a further reduction in the dose to the cochlea and oral cavity. With nine and seven fields, the dose to the contra-lateral parotid gland was increased, but this was avoided by optimisation of the beam directions. The benefits of IMRT were maintained with three or four fields when the beam angles were optimised, but were also achieved using a four-field class solution. Clinical trials are required to confirm the clinical benefits of these improved dose distributions.

Antioxidants and periodontitis in 60-70 year- old men

Objective: The aim was to investigate the association between periodontal health and the serum levels of various antioxidants including carotenoids, retinol and vitamin E in a homogenous group of Western European men.
Materials and Methods: A representative sample of 1258 men aged 60-70 years, drawn from the population of Northern Ireland, was examined between 2001 and 2003. Each participant had six or more teeth, completed a questionnaire and underwent a clinical periodontal examination. Serum lipid-soluble antioxidant levels were measured by high-performance liquid chromatography with diode array detection. Multivariable analysis was carried out using logistic regression with adjustment for possible confounders. Models were constructed using two measures of periodontal status (low- and high-threshold periodontitis) as dependent variables and the fifths of each antioxidant as a predictor variable.
Results: The levels of a- and ß-carotene, ß-cryptoxanthin and zeaxanthin were highly significantly lower in the men with low-threshold periodontitis (p<0.001). These carotenoids were also significantly lower in high-threshold periodontitis. There were no significant differences in the levels of lutein, lycopene, a- and ?-tocopherol or retinol in relation to periodontitis. In fully adjusted models, there was an inverse relationship between a number of carotenoids (a- and ß-carotene and ß-cryptoxanthin) and low-threshold periodontitis. ß-Carotene and ß-cryptoxanthin were the only antioxidants that were associated with an increased risk of high-threshold severe periodontitis. The adjusted odds ratio for high-threshold periodontitis in the lowest fifth relative to the highest fifth of ß-cryptoxanthin was 4.02 (p=0.003).
Conclusion: It is concluded that low serum levels of a number of carotenoids, in particular beta-cryptoxanthin and beta-carotene, were associated with an increased prevalence of periodontitis in this homogenous group of 60-70-year-old Western European men.

Autoradiographic and scintillation analysis of 5-aminolevulinic acid permeation through epithelialised tissue: Implications for topical photodynamic therapy of superficial gynaecological neoplasias

Purpose. Aminolevulinic acid (5-ALA) diffusion through both keratinised and non-keratinised tissue, used as a model tissue substrates, was evaluated, together with the depth of permeation and the concentration achieved following delivery from bioadhesive patch and proprietary cream formulations. Materials and Methods. Moisture-activated, bioadhesive patches loaded with 5-ALA at concentrations of 19.0, 38.0 and 50.0 mg cm(-2) and an o/w cream (20% w/w 5-ALA) were radiolabelled with C14 5-ALA and applied to excised human vaginal tissue and porcine skin. After 1, 2 and 4 h, tissue was sectioned in two orientations and the 5-ALA concentration at specific depths determined using autoradiography and liquid scintillation counting (LSC). Results. The stratum corneum was a significant barrier to 5-ALA permeation, with concentrations in tissue dependent on application time and drug loading. 5-ALA was detected at 6 mm using autoradiography after 2 h, with LSC showing phototoxic concentrations at 2.375 mm after 4 h of application. Inclusion of oleic acid and dimethyl sulphoxide in bioadhesive patches increased 5-ALA significantly in neonate porcine tissue, but only for patches cast from blends containing 5% w/w oleic acid. Conclusions. The bioadhesive patch described delivered 5-ALA to depths of at least 2.5 mm in tissue types indicative of vulval skin, suggesting that photodynamic therapy of deep vulval intraepithelial neoplasia is feasible using this means of bioadhesive 5-ALA delivery.

In Vivo Age Dependency of Unfractionated Heparin in Infants and Children

Introduction
Unfractionated Heparin (UFH) is used widely in paediatrics. Paediatric specific recommendations for UFH therapy are few, with the majority of recommendations being extrapolated from adult practice. In vitro studies have shown that this practice may be suboptimal. This study aimed to improve the understanding of the impact of age upon UFH response in vivo.

Materials and Methods
This prospective, observational study, conducted in the Paediatric Intensive Care Unit (PICU), included: patients 16 years or younger; treated with UFH of at least 10 U/Kg/hr. Laboratory analysis included: Antithrombin, APTT, Anti-Xa, Anti-IIa and thrombin generation expressed as the Endogenous Thrombin Potential. Results were grouped according to patient age (i.e. < 1, 1-5, 6-10 and 11-16 years).

Results
85 patients received an equivalent mean UFH dose with a median duration of 3 days. Antithrombin levels were decreased compared to age-related norms in children up to 11 years of age. APTT results were comparable across the age-groups. The Anti-Xa results using two different assays showed a trend for lower values in younger children. All children less than one year old recorded Anti-Xa values outside the therapeutic range for heparin therapy, for both assays. There was a trend for decreased Anti-IIa activity in younger children. Endogenous Thrombin Potential showed a significant trend for increased inhibition in older children. In vitro Antithrombin supplementation did not change the Anti-Xa or thrombin generation.

Conclusions
This study confirms that, in vivo, for the same dose of UFH, the anti Xa and anti IIa effect, as well as the inhibition of endogenous thrombin potential is age dependent and that these differences are not purely AT dependent. The implication is that the anticoagulant and antithrombotic effect of a given dose of UFH differs with age. Clinical outcome studies to determine the optimal dosing for each age group are warranted.

Abbreviations
UFH, Unfractionated Heparin; ETP, Endogenous Thrombin Potential; AT, Antithrombin; APTT, Activated Partial Thromboplastin Time

Psychological status of patients referred for orthognathic correction of skeletal II and III discrepancies

Objective: To establish the extent of psychological problems among patients who require orthognathic treatment. Materials and Methods: Five aspects of psychological functioning were assessed for 162 patients who required orthognathic treatment and compared with 157 control subjects.

Results: Analysis of variance did not detect any significant difference in the five psychological scores recorded for the skeletal II, skeletal III, and control groups. The proportion of subjects with one or more psychological measure beyond the normal range was 27% for skeletal II subjects, 25% for skeletal III subjects, and 26% for control subjects. One skeletal II subject (1.5%), three skeletal III subjects (3%), and five control subjects (3%) required referral for psychological counseling.

Conclusions: The orthognathic patients did not differ significantly from the control subjects in their psychological status. © 2010 by The EH Angle Education and Research Foundation, Inc.

Targeting death receptors in bladder, prostate and renal cancer

PURPOSE: We describe key components of normal and aberrant death receptor pathways, the association of these abnormalities with tumorigenesis in bladder, prostate and renal cancer, and their potential application in novel therapeutic strategies targeted toward patients with cancer.

MATERIALS AND METHODS: A MEDLINE literature search of the key words death receptors, TRAIL (tumor necrosis factor related apoptosis inducing ligand), FAS, bladder, prostate, renal and cancer was done to obtain information for review. A brief overview of the TRAIL and FAS death receptor pathways, and their relationship to apoptosis is described. Mechanisms that lead to nonfunction of these pathways and how they may contribute to tumorigenesis are linked. Current efforts to target death receptor pathways as a therapeutic strategy are highlighted.

RESULTS: Activation of tumor cell expressing death receptors by cytotoxic immune cells is the main mechanism by which the immune system eliminates malignant cells. Death receptor triggering induces a caspase cascade, leading to tumor cell apoptosis. Receptor gene mutation or hypermethylation, decoy receptor or splice variant over expression, and downstream inhibitor interference are examples of the ways that normal pathway functioning is lost in cancers of the bladder and prostate. Targeting death receptors directly through synthetic ligand administration and blocking downstream inhibitor molecules with siRNA or antisense oligonucleotides represent novel therapeutic strategies under development.

CONCLUSIONS: Research into the death receptor pathways has demonstrated the key role that pathway aberrations have in the initiation and progression of malignancies of the bladder, prostate and kidney. This new understanding has resulted in exciting approaches to restore the functionality of these pathways as a novel therapeutic strategy.

KCNQ Currents and Their Contribution to Resting Membrane Potential and the Excitability of Interstitial Cells of Cajal From the Guinea Pig Bladder

PURPOSE: The presence of novel KCNQ currents was investigated in guinea pig bladder interstitial cells of Cajal and their contribution to the maintenance of the resting membrane potential was assessed. MATERIALS AND METHODS: Enzymatically dispersed interstitial cells of Cajal were patch clamped with K(+) filled pipettes in voltage clamp and current clamp modes. Pharmacological modulators of KCNQ channels were tested on membrane currents and the resting membrane potential. RESULTS: Cells were stepped from -60 to 40 mV to evoke voltage dependent currents using a modified K(+) pipette solution containing ethylene glycol tetraacetic acid (5 mM) and adenosine triphosphate (3 mM) to eliminate large conductance Ca activated K channel and K(adenosine triphosphate) currents. Application of the KCNQ blockers XE991, linopirdine (Tocris Bioscience, Ellisville, Missouri) and chromanol 293B (Sigma) decreased the outward current in concentration dependent fashion. The current-voltage relationship of XE991 sensitive current revealed a voltage dependent, outwardly rectifying current that activated positive to -60 mV and showed little inactivation. The KCNQ openers flupirtine and meclofenamic acid (Sigma) increased outward currents across the voltage range. In current clamp mode XE991 or chromanol 293B decreased interstitial cell of Cajal resting membrane potential and elicited the firing of spontaneous transient depolarizations in otherwise quiescent cells. Flupirtine or meclofenamic acid hyperpolarized interstitial cells of Cajal and inhibited any spontaneous electrical activity. CONCLUSIONS: This study provides electrophysiological evidence that bladder interstitial cells of Cajal have KCNQ currents with a role in the regulation of interstitial cell of Cajal resting membrane potential and excitability. These novel findings provide key information on the ion channels present in bladder interstitial cells of Cajal and they may indicate relevant targets for the development of new therapies for bladder instability.

Advanced glycation end products cause increased CCN family and extracellular matrix gene expression in the diabetic rodent retina

Aims/hypothesis: Referred to as CCN, the family of growth factors consisting of cystein-rich protein 61 (CYR61, also known as CCN1), connective tissue growth factor (CTGF, also known as CCN2), nephroblastoma overexpressed gene (NOV, also known as CCN3) and WNT1-inducible signalling pathway proteins 1, 2 and 3 (WISP1, -2 and -3; also known as CCN4, -5 and -6) affects cellular growth, differentiation, adhesion and locomotion in wound repair, fibrotic disorders, inflammation and angiogenesis. AGEs formed in the diabetic milieu affect the same processes, leading to diabetic complications including diabetic retinopathy. We hypothesised that pathological effects of AGEs in the diabetic retina are a consequence of AGE-induced alterations in CCN family expression.

Materials and methods: CCN gene expression levels were studied at the mRNA and protein level in retinas of control and diabetic rats using real-time quantitative PCR, western blotting and immunohistochemistry at 6 and 12 weeks of streptozotocin-induced diabetes in the presence or absence of aminoguanidine, an AGE inhibitor. In addition, C57BL/6 mice were repeatedly injected with exogenously formed AGE to establish whether AGE modulate retinal CCN growth factors in vivo.

Results: After 6 weeks of diabetes, Cyr61 expression levels were increased more than threefold. At 12 weeks of diabetes, Ctgf expression levels were increased twofold. Treatment with aminoguanidine inhibited Cyr61 and Ctgf expression in diabetic rats, with reductions of 31 and 36%, respectively, compared with untreated animals. Western blotting showed a twofold increase in CTGF production, which was prevented by aminoguanidine treatment. In mice infused with exogenous AGE, Cyr61 expression increased fourfold and Ctgf expression increased twofold in the retina.

Conclusions/interpolation: CTGF and CYR61 are downstream effectors of AGE in the diabetic retina, implicating them as possible targets for future intervention strategies against the development of diabetic retinopathy.